Cells and Methodology to Generate Non-Segmented Negative-Strand RNA Viruses

ABSTRACT

The present invention relates to recombinant cells as well as to methods for the generation of non-segmented negative-sense single-stranded RNA viruses (NNV or mononegavirales) from cloned deoxyribonucleic acid (cDNA), especially from measles virus and in particular from attenuated strains such as those approved for vaccination, in particular from the attenuated Schwarz measles virus and various recombinant Schwarz measles-based viruses expressing heterologous sequences. Such rescued viruses can be used, after amplification, as vaccines for immunization against measles and/or against the heterologous peptides or proteins expressed.

The present invention relates to recombinant cells as well as to methods for the generation of non-segmented negative-sense single-stranded RNA viruses (NNV or mononegavirales) from cloned deoxyribonucleic acid (cDNA), especially from measles virus and in particular from attenuated strains such as those approved for vaccination, in particular from the attenuated Schwarz measles virus and various recombinant Schwarz measles-based viruses expressing heterologous sequences. Such rescued viruses can be used, after amplification, as vaccines for immunization against measles and/or against the heterologous peptides or proteins expressed.

Live attenuated RNA viruses make very efficient vaccines. Among these, measles vaccine has been used in hundreds of millions of children and has been proven to be effective and safe. This vaccine induces life-long immunity after one or two injections. It is easily produced on a large scale at low cost in most countries. These advantages make measles virus, especially attenuated vaccine strains, a good candidate vector to immunize children but even in some circumstances adult populations, against both measles and/or other infectious pathologies, especially viral pathologies such as AIDS (retroviruses), flavivirus or coronavirus (SARS) diseases.

Live attenuated measles virus has been used as vaccine since the 1960s and is one of the most effective and safest human vaccines. Vaccination campaigns have been very effective to control measles in developed countries. However, due to inadequate distribution of the vaccine in developing countries, measles still infects approximately 45 million individuals and is responsible for the death of 700,000 children per year. The WHO has therefore stepped up its global vaccination program for the next 10-20 years (C.D.C., 2005). Taking advantage of the WHO campaigns, the use of vaccine vectors derived from measles vaccine would allow in certain regions of the world the simultaneous immunization of children against measles and other infectious diseases with new multivalent, especially bivalent pediatric vaccines that are both safe and effective.

Measles virus (MV) belongs to the genus Morbillivirus in the family Paramyxoviridae. It is an enveloped virus with a non-segmented RNA genome of negative polarity (15,894 bp). Measles can only be contracted once as the immune system mounts a strong specific response and establishes life-long memory protecting against re-infection. Such protection is based on both the production of antibodies and memory cytotoxic CD8⁺ T lymphocytes (CTL). Pathogenic strains strongly disrupt hematopoiesis (Arneborn et al., 1983; Kim et al., 2002; Okada et al., 2000) thus resulting in transitory immunosuppression responsible for most deaths due to measles infection in developing countries. In contrast to primary strains, attenuated strains do not induce immunosuppression (Okada et al., 2001).

The Edmonston strain of measles virus was isolated in 1954 by culture in primary human cells (Enders et al., 1954). Adaptation to chicken embryonic fibroblasts produced vaccine seeds that were furthermore attenuated by subsequent passages in chicken embryonic fibroblasts (Schwarz et al., 1962). The Schwarz and Moraten strains that possess identical nucleotide sequences (Parks et al., 2001a; Parks et al., 2001b) constitute the most frequently used measles vaccine. Vaccination with one or two injections induces life-long immunity (Griffin et al., 2001; Hilleman et al., 2002). Persistence of CD8 cells and antibodies has been demonstrated up to 25 years after vaccination (Ovsyannikova et al., 2003). The measles vaccine is easily produced on a large scale in most countries and may be made available at low cost. Attenuation of the viral genome results from an advantageous combination of multiple mutations. Thus, the vaccine is very stable and reversion of vaccine strains has never been observed to date (Hilleman et al., 2002). In addition, the virus replicates only in the cytoplasm, eliminating any risk of integrating into chromosomes of the host genome. These features make live attenuated measles vaccine an excellent candidate for the development of a multivalent vaccine vector. To this end, an infectious cDNA corresponding to the Edmonston B MV strain antigenome has been cloned and a reverse genetics technique enabling the production of the corresponding virus was established (Radecke et al., 1995).

The inventors have previously developed a vector using the Schwarz M V, the most commonly used measles vaccine in the world (Combredet et al., 2003). This vector can stably express a variety of genes or combination of large genes for more than 12 passages. Recombinant MV vectors containing 4,000-5,000 additional nucleotides were produced, representing an additional 30% of genome. These viruses were produced in cell culture at titers comparable to standard MV. After 12 passages and an amplification factor of 10²⁰, more than 96% of the infected cells continued to express the additional genes. This remarkably stable expression, also observed for other members of the Mononegavirales (Schnell et al., 1996) is likely due to the absence of geometric constraints on the size of the genome by these helicoid nucleocapsid viruses, in contrast to viruses with icosahedral capsids. Moreover, MV infects cells of the immune system (macrophages and dendritic cells), thus delivering the cargo antigens directly to the most effective antigen presenting cells, a major advantage for a vaccine vector. Finally, the MV genome is small, thus avoiding the response to the vector overwhelming the response to transgenes.

Based on the assumption that the safety and efficacy of an attenuated strain ultimately depends on its genome sequence, the inventors cloned the infectious cDNA corresponding to the antigenome of the Schwarz/Moraten measles virus from virus particles purified from an industrial preparation of the Schwarz vaccine with optimal procedures to maintain fidelity (Combredet et al., 2003). To optimize the output of the reverse genetics system, the antigenomic viral cDNA was placed under the control of the T7 phage RNA polymerase promoter with an additional GGG motif required for optimal efficacy. To allow exact cleavage of the viral RNA, a hammerhead ribozyme was inserted between the GGG motif and the first viral nucleotide, and the ribozyme from hepatitis delta virus was placed downstream of the last viral nucleotide. The resulting pTM-MVSchw plasmid enabled the production of the corresponding virus using a previously described reverse genetics system based on the transfection of human helper cells (Radecke et al., 1995). To prevent adaptation of the recombinant vaccine to non-certified cells, the helper cells transfected with cDNA were co-cultivated with chicken embryonic fibroblasts, the cells in which the virus was originally selected and in which it is currently produced. After several passages of recombinant virus the sequence of its entire genome was found identical to the original sequence (Combredet et al., 2003). The immunogenicity of the virus rescued from pTM-MVSchw plasmid was evaluated in transgenic mice and macaques and compared with the industrially manufactured Schwarz vaccine. All vaccinated macaques developed anti-MV antibodies and specific cellular responses. No differences were observed between the Schwarz virus produced from the cDNA and the original vaccine, indicating that the cloned virus had the same immunogenicity as the parental vaccine (Combredet et al., 2003). This molecular clone allows the production of the Schwarz measles vaccine without depending on the seeding stocks.

The pTM-MVSchw plasmid was modified for the expression of foreign genes by the introduction of additional transcriptional units (ATU) at different positions of the genome. These ATUs are multi-cloning site cassettes inserted for example in a copy of the intergenic N-P region of the viral genome (containing the cis acting sequences required for transcription). The enhanced green fluorescent protein (eGFP) gene was inserted into this cassette. The ATU was introduced into the pTM-MVSchw plasmid in two positions (between the P and M genes and between the H and L genes). Irrespective of the additional sequence, the total number of antigenomic nucleotides must be maintained as a multiple of six to fulfill the “rule of 6 nucleotides” that optimizes viral replication (Calain et al., 1993). The GFP transgene was expressed in all infected cell types, confirming that the recombinant Schwarz measles virus works as a vector. This vector allows the design of combined vaccines based on a live attenuated approved vaccine strain that is currently globally in use. This work is the object of international application WO 2004/000876 which is incorporated herewith by reference.

The use of such MV-based live recombinant vaccines at large scale depends on the possibility of growing them stably and at good titers on certified cells (such as primary chicken embryonic fibroblasts (CEF) or human diploid MRC5). These cells usually produce MV at moderate titers as compared to laboratory cell lines, such as African green monkey Vero cells, that produce at high titers. Thus, the initial seed must be obtained at a relatively high titer. This initial seed is produced from cDNA by reverse genetics.

While positive-strand RNA or DNA viruses can be easily obtained in vitro after transfection of their engineered infectious cDNA or DNA in appropriate cells, the negative-strand RNA viruses cannot be rescued directly by reverse genetics from their cDNA. The genome of negative-strand RNA viruses is not able to initiate in vitro an infectious cycle because it does not code directly for proteins. Both transcription and replication require a transcriptase-polymerase enzymatic complex contained in the nucleoproteins encaspidating the viral genome (RNP complex). Thus, the generation of recombinant negative-strand RNA viruses from cDNA involves reconstitution of active RNPs from individual components: RNA and proteins (Fields B. N. et al—Lippincott Raven publishers 1996, p. 1953-1977).

For the last 15 years, a remarkable set of work from numerous laboratories has allowed the establishment of different systems for rescuing almost all negative-strand RNA viruses from their cDNA (for review see Conzelmann). In contrast to the viruses with segmented genomes, the RNPs of non-segmented negative-strand RNA viruses (Mononegavirales) are tightly structured and contain, in addition to the nucleoprotein (N), the assembly and polymerase cofactor phosphoprotein (P) and the viral RNA polymerase large protein (L), The first infectious Mononegavirales, the rabies rhabdovirus, was recovered from cDNA in 1994 (Schnell et al. 1994). The approach involved intracellular expression of rabies virus N, P, and L protein, along with a full-length RNA whose correct 3′ end was generated by the hepatitis delta virus (HDV) ribozyme. A transcript corresponding to the viral antigenome (positive strand) rather than to the genome (negative strand) was used to avoid an antisense problem raised by the presence of N, P, and L sequences in full-length RNAs. In this system, the essential helper proteins were provided by a replication-competent vaccinia vector encoding the phage T7 RNA polymerase to drive T7-specific transcription of plasmids encoding the required N, P and L proteins. Similar systems allowed recovery of infectious rabies viruses (Schnell et al. 1994; Ito et al. 2001), VSV (Lawson et al. 1995; Whelan et al. 1995), as well as the Paramyxoviridae Sendai virus (Garcin et al. 1995; Kato et al. 1996; Leyrer et al. 1998; Fujii et al. 2002), HP1V-3 (Hoffman and Banerjee 1997) and measles virus (Takeda et al. 2000; Fujii et al. 2002).

To avoid the use of replication-competent vaccinia, which requires that the rescued virus be separated from helper virus, several non-replicative helper viruses have been adapted to provide helper proteins to rescue non-segmented negative-strand RNA viruses. The highly attenuated modified vaccinia virus Ankara (MVA) expressing T7 RNA polymerase has been used for recovery of the Pneumovirus RSV (Collins et al. 1995), the Rubulavirus, SV5 (He et al. 1997), HPIV-3 (Durbin et al. 1997), rinderpest virus (Baron and Barrett 1997), and measles virus (Schneider et al. 1997), mumps virus (Clarke et al. 2000), CDV (Gassen et al. 2000), HPIV-2 (Kawano et al. 2001), and BPIV-3 (Schmidt et al. 2000). A recombinant fowlpox virus expressing the T7 RNA polymerase has been used for the recovery of the avian Paramyxoviridae NDV (Peeters et al. 1999) and of a chimeric rinderpest virus (Das et al. 2000).

To rescue Mononegavirales without contamination by any infectious or defective viral vector, cell lines expressing T3 or T7 RNA polymerase have been generated. In this case, in the absence of RNA-capping activity in the cytoplasm, protein expression was achieved using the IRES from encephalomyocarditis virus (EMCV) located upstream of the coding regions. A human embryo kidney cell line (293-3-46) expressing T7 RNA polymerase and measles virus proteins N and P was established to recover the Edmonston vaccine strain of measles virus (Radecke et al. 1995). The virus was rescued after transfection of plasmids specifying MV antigenomic RNA and L mRNA. It was shown that rescue efficiency in these cells, which was very low initially, was increased by heat shock treatment of the transfected cultures and additional cocultivation of transfected cells on Vero cells (Parks et al., 1999). Another cell line expressing T7 RNA polymerase (BSR T7/5) and based on baby hamster kidney cells (BHK) was used for recovery of BRSV (Buchholz et al. 2000), rabies viruses (Finke and Conzelmann 1999), VSV (Harty et al. 2001), NDV (Romer-Oberdorfer et al. 1999), and Ebola virus (Volchkov et al. 2001).

The inventors have used the 293-3-46 cell line to rescue the Schwarz vaccine MV vector (Combredet et al., 2003). However, they have experienced that, even using the heat shock method on transfected cells (Parks et al., 1999) and their cocultivation on Vero or CEF cells, the rescue was rather irreproducible and still at very low yield, or even impossible for some recombinants containing large additional sequences. This was due to the instability of helper cells since it was observed that the efficiency depends on the number of their passages. These cells have been generated by selecting geneticin-resistant clones of 293 cells transfected with pSC6-N, pSC6-P and pSC6-T7-NEO encoding respectively the MV N and P genes and the T7 RNA polymerase gene under control of the CMV promoter and a neomycin resistance gene (Radecke et al., 1995). The stability of their activity depends on their continuous selection under geneticin (G-418), and the removing of antibiotic during transfection and rescue experiments. During the illicit plasmid-based recombination of foreign DNA into chromosomic DNA, the concatemeres formed by plasmids are recombined and the geneticin selection maintains only the individual copies, which are very few. This might explain the reduction of efficiency observed with 293-3-46 cells after a few passages.

Therefore, there exists a need in the art for a new method for generating helper cell lines able to rescue, reproducibly and with high efficiency, recombinant, non-segmented negative-strand RNA viruses from cDNA, optionally modified, and without contamination by any other helper virus such as vaccinia virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: schematic representation of the plasmids HIV-1-TRIPΔU3.CMV-T7 (A), HIV-1-TRIPΔU3.CMV-nlsT7 (B), HIV-1-TRIPΔU3.CMV-N (C) and HIV-1-TRIPΔU3.CMV-P (D). ψ: packaging psi motif: RRE: Rev-responsive element; cPPT: central polypurine tract, CTS: central termination sequence, CMVie: cytomegalovirus immediate-early promoter; ΔU3: deletion of parts of U3.

FIG. 2: Western blot showing the expression of the MV N and P proteins in different cell lysates; (A) non transduced 293T, previously described 293-3-46 cell line at two different passages (17 and 19), 293nlsT7-NP and 293T7-NP cell populations generated after transduction with lentiviral vectors; (B) MV-infected Vero cells, 293-3-46 cell line at two different passages (17 and 27), eight 293T7-NP cell clones; (C) MV-infected Vero cells, 293-3-46 cell line (passage 17), eight 293nlsT7-NP cell clones, uninfected Vero cells. Blots were probed with anti-MV NP antibody (1/500) and HRP anti-mouse Ig secondary antibody (1/1000).

BRIEF DESCRIPTION OF THE SEQUENCES

Nucleotide sequences of various retrovirus DNA FLAP are defined in different viruses: CAEV (SEQ ID NO:1), EIAV (SEQ ID NO:2), VISNA (SEQ ID NO:3), SIV AGM (SEQ ID NO:4), HIV-2 ROD (SEQ ID NO:5), HIV-1 LAI (SEQ ID NO:6) and HIV-1 (SEQ ID NO:7). The nucleotide sequences of the T7 RNA polymerase, the nls T7 RNA polymerase and the N, P and L proteins of the MV virus are defined respectively in SEQ ID NO: 8, 10, 12, 14 and 16, as well as their respective corresponding protein sequences in SEQ ID NO: 9, 11, 13, 15 and 17. The complete nucleotide sequence of the pTM-MVSchw plasmid (CNCM 1-2889) is defined in SEQ ID NO: 18. The complete nucleotide sequence of the pEMC-LSchw plasmid (CNCM 1-3881) is defined in SEQ ID NO: 19.

DETAILED DESCRIPTION

The present invention relates to a cell stably producing at least a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a phosphoprotein (P) of a non-segmented negative-strand RNA virus, or functional derivatives thereof. In a particular embodiment, the cell of the invention stably produces a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a phosphoprotein (P) of a non-segmented negative-strand RNA virus, or functional derivatives thereof.

The cells of the present invention are recombinant cells meaning that these cells are the results of in vitro purposive genetic manipulation resulting in recombination of genomic sequences of the cells with heterologous sequences, i.e., sequences originating from a different cell or organism. Starting from isolated cells, recombinant cells are prepared, have genetic and/or phenotypic features different from those of the starting cells, and also provide for the stable expression or production of at least a RNA polymerase, the N protein and the P protein of one or several non-segmented negative-strand RNA viruses. Cells of the invention are claimed as product, external to the body of a human being.

The expression “stably producing” means that cells express or produce at least the RNA polymerase, the N protein and the P protein over a number of cell divisions equals to or higher than about 65, advantageously as long as the cell survives. According to a particular embodiment of the invention, the recombinant cells express or produce the at least three proteins, i.e., at least the RNA polymerase, the N protein and the P protein, continually in time. According to a particular embodiment of the invention, the integrity, i.e., the primary amino acid sequence, of these three proteins is maintained, ensuring that the proteins expressed or produced are always the same.

The stable production of the RNA polymerase, the N protein and the P protein is independent of the presence in the cell, of plasmids) carrying the coding sequence of these proteins. Therefore, even though plasmids may be used at a particular step of the in vitro or ex vivo cell manipulation, the resulting recombinant cells, which stably produce the three or the at least three proteins, do not contain plasmids anymore. In that way, the expression is said plasmid-independent, in contrast to recombinant cells in which protein expression is driven by plasmid(s).

In a particular embodiment of the invention, the stable expression of the RNA polymerase, of the N protein and of the P protein, does not require the presence of a drug, such as an antibiotic, i.e., the stable expression does not require a selection pressure. Therefore, the stable production does not require the mandatory presence of plasmid(s) for survival, said plasmid bearing the coding sequence of the protein(s) to express.

Another feature of the invention is that each of the at least three proteins, i.e., at least the RNA polymerase, the N protein and the P proteins are produced or expressed at similar level over time. “Similar level” as used herein means that the expression of each of the three proteins is steady during the cell life, even after cell division, with a variation in expression level which is not more than about 30%, particularly not more than about 20% and preferably not more than about 10%, as compared to mean expression calculated at different times of the cell life.

The RNA polymerase expressed or produced by the cells of the invention is any polymerase suitable for synthesizing non-segmented negative-sense single-stranded viral RNA (vRNA) derived from a cDNA clone, in a rescue system. The nature of the polymerase depends essentially on the nature of the RNA promoter polymerase sequence located in the cDNA clone of the non-segmented negative-strand single-stranded RNA virus, used for the rescue system (also called reverse genetics, or de novo synthesis of negative-sense RNA viruses from cloned cDNA). As an example, the RNA polymerase is the T7 phage RNA polymerase, or its nuclear form (nlsT7).

The expressions “N protein” and “protein” refer respectively to the nucleoprotein (N) of a non-segmented negative-strand single-stranded RNA virus and the phosphoprotein (P) of a non-segmented negative-strand single-stranded RNA virus. Examples of families subfamilies, genius or species of non-segmented negative-strand single-stranded RNA viruses from which the N and/or P protein can be derived are listed in Table 1.

In a particular embodiment, the N and P proteins of a non-segmented negative-strand RNA virus are from the same virus, either from the same virus strain or from different virus strains. In another embodiment, the N and P proteins of a non-segmented negative-strand RNA virus are from different non-segmented negative-strand RNA virus.

TABLE 1 Family, subfamily, genus and species of several non-segmented negative-strand RNA viruses (NNV) of the order Mononegavirale. Family Subfamily Genus Species Abbreviation Rhabdoviridae / Vesiculovirus Vesicular stomatitis VSV virus Lyssavirus Rabies virus RV Paramyxoviridae Paramyxovirinae Morbillivirus Measles virus MV Rinderpest virus RPV Canine distemper CDV virus Respirovirus Sendai virus SeV Human parainfluenza hPIV3 virus type 3 Bovine parainfluenza bPIV3 virus type 3 Rubulavirus Simian virus type 5 SV5 Mumps virus Human parainfluenza hPIV2 virus type 2 Newcastle disease NDV virus Pneumovirinae Pneumovirus Human respiratory hRSV syncytial virus Bovine respiratory bRSV syncytial virus Filoviridae / Ebola-like Ebola virus / viruses

In particular embodiment, the N and P proteins are derived from a Mononegavirus, preferably a Paramyxoviridae virus, preferably a Paramyxovirinae virus, and most preferably a Morbillivirus virus. As an example of Morbillivirus is the Measles virus (MV), in particular an attenuated non immunosuppressive strain, e.g. an approved strain for a vaccine, and especially the Schwarz MV strain or the Edmonston (Ed) strain or a derivative from these strains. An approved strain for a vaccine is defined by the FDA (US Food and drug administration) as having the following provisions: safety, efficacy, quality and reproducibility, after rigorous reviews of laboratory and clinical data (www.fda.gov/cber/vaccine/vacappr.htm).

Each time reference is made in the present application, to non-segmented negative strand RNA virus, it possibly applies in particular to the specific viruses listed herein, and especially to a measles virus, in particular to the Schwarz strain.

The expression “functional derivatives thereof” refers to any functional variants including fragments of the RNA polymerase and/or the N protein and/or the P protein, provided that the functional derivatives keep the activity of the protein they are derived from, at least as a ribonucleoprotein complex (RNP complex), functional in transcription and replication in a virus genome, in a rescue system enabling the production of non-segmented negative-sense RNA viruses from cloned cDNA.

A functional variant is defined by a nucleic acid encoding said functional variant proteins, having at least one of the following features:

-   -   the nucleic acid encoding the functional variant hybridizes in         high stringency conditions with a nucleic acid encoding the         wild-type (reference) RNA polymerase or with the N protein and         the P protein of an identified non-segmented negative-strand RNA         strain or virus. High stringency conditions are defined by         Sambrook et al. in Molecular Cloning: a laboratory manual         (1989). These conditions of high stringency encompass: use a         prewashing solution for the nitrocellulose filters 5×SSC, 0.5%         SDS, 1.0 mM EDTA (pH 8.0), hybridisation conditions of 50%         formamide, 6×SSC at 42° C. and washing conditions at 68° C.,         0.2×SSC and 0.1% SDS. Protocols are known to those having         ordinary skill in the art. Moreover, the skilled artisan will         recognize that the temperature and wash solution salt         concentration can be adjusted as necessary according to         experimental constraints;     -   the nucleic acid encoding the functional variant presents at         least 80%, preferably 90%, more preferably 95% or even 99%         similarity with a native nucleic acid encoding the RNA         polymerase, the N protein or the protein, said similarity being         calculated over the entire length of both sequences;     -   the nucleic acid encoding the functional variant differs from a         native nucleic acid encoding the RNA polymerase, the N protein         or the P protein by at least one nucleotide substitution,         preferably 1, 2, 3, 4 or 5 substitution(s), optionally         conservative substitutions (nucleotide substitution(s) not         altering the amino acid sequence), by at least one nucleotide         deletion or addition, preferably 1, 2, 3, 4 or 5 nucleotide(s)         deletion or addition.     -   A fragment is defined in the present application as a part of         the full-length RNA polymerase, of the N protein or of the P         protein, as long as the fragment has the same activity as the         entire protein from which it is derived, at least as a         ribonucleoprotein complex (RNP complex) as disclosed herein. In         a particular embodiment, the fragment represents at least 70%,         particularly 80%, and more particularly 90% or even 95% of the         full-length protein.

Accordingly, where reference is made herewith to RNA polymerase, N or P proteins or to their coding sequences, the description similarly applies to their functional derivatives as defined herein.

According to a particular embodiment a recombinant cell of the invention comprises, integrated in its genome, at least one copy of a nucleic acid encoding a RNA polymerase, at least one copy of a nucleic acid encoding a N protein of a non-segmented negative-strand RNA virus, and at least one copy of a nucleic acid encoding a P protein of a non-segmented negative-strand RNA virus. Optionally, the nucleic acids encoding the three proteins above are, each or at least one of these, under the control of transcription regulatory element(s) The expression “integrated in the genome” means that the at least one copy of a nucleic acid under the control of transcription regulatory element(s) is located within the genome of the recombinant cells, under conditions enabling said cells to stably express the protein encoded by the nucleic acid. In a particular embodiment, the recombinant cell of the invention comprises further, integrated in its genome, at least one copy of a nucleic acid encoding a L protein of a non-segmented negative-strand RNA virus.

“at least one copy” means that the nucleic acid encoding the RNA polymerase and/or the N protein and/or the P protein and/or the L protein may be present in one or several copies, preferably exactly or at least 2, 3, 4, 5, 6, 7, 8, 9, 10 copies, or more, depending on the expression level required for each of these proteins.

In a particular embodiment of the invention, the cells of the invention also contain at least one copy of a DNA flap integrated into the cell genome. A DNA flap is a nucleotide sequence of retroviral especially lentiviral, or retroviral-like origin comprising two essential regions, i.e., the cPPT (central polypurine tract) and the CTS (cis-acting termination region) regions, wherein the cPPT and CTS regions induce a three-stranded DNA structure during replication of DNA containing them (previously defined in Zennou et al., 2000; and in applications WO99/55892 and WO01/27300). In a particular embodiment, the DNA flap is inserted immediately upstream of the internal promoter enabling transcription of the nucleic acids encoding the RNA polymerase, the N protein, the P protein and possibly the L protein. The DNA flap (cPPT-CTS) is inserted into the retroviral-derived vectors of the invention in a functional orientation i.e., the cPPT region is in 5′ with respect to the CTS region (the 5′ part of the vector corresponding to the LTR containing the primer binding site (PBS), and the 3′ part of the vector corresponding to the region containing the 3′PPT).

A DNA flap suitable for the invention may be obtained from a retrovirus especially from a lentivirus or retrovirus-like organism such as retrotransposon, prepared synthetically (chemical synthesis) or by amplification of the DNA flap from any retrovirus especially from a lentivirus nucleic acid such as by Polymerase chain reaction (PCR). The DNA flap may be obtained from a retrovirus, especially a lentivirus, especially a human retrovirus or lentivirus and in particular a HIV retrovirus, the CAEV (Caprine Arthritis Encephalitis Virus) virus, the EIAV (Equine Infectious Anaemia Virus) virus, the VISNA virus, the SIV (Simian Immunodeficiency Virus) virus or the FIV (Feline Immunodeficiency Virus) virus. In a more preferred embodiment, the DNA flap is obtained from an HIV retrovirus, for example HIV-1 or HIV-2 virus or any different isolate of these two types.

Preferred DNA flap comprises or consists in the sequences as defined in SEQ ID NO: 1 to 7. It is noteworthy that the DNA flap is used isolated from its natural (viral genome) nucleotide context i.e., isolated from the pol gene in which it is naturally contained in a lentivirus. Therefore, the DNA flap is used, in the present invention, deleted from the unnecessary 5′ and 3′ parts of the pol gene and is recombined with sequences of different origin. According to a particular embodiment, a DNA flap has a nucleotide sequence of about 90 to about 150 nucleotides, in particular from about 100 to about 140 nucleotides.

The invention also concerns a cell obtainable by recombination of its genome with (1) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a RNA polymerase, (2) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a N protein of a non-segmented negative-strand RNA virus, and (3) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a P protein of a non-segmented negative-strand RNA virus. Definitions given above apply to these cells.

The invention is also directed to a cell obtainable by recombination of its genome with (1) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a RNA polymerase, (2) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a N protein of a non-segmented negative-strand RNA virus, (3) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a P protein of a non-segmented negative-strand RNA virus and (4) an expression vector comprising a DNA flap, and at least one copy of a nucleic acid encoding a L protein of a non-segmented negative-strand RNA virus. Definitions given above apply to these cells. Definitions given above apply to these cells.

The invention encompasses a cell obtainable by recombination of its genome with an expression vector comprising at least one copy of a nucleic acid encoding a RNA polymerase, at least one copy of a nucleic acid encoding a N protein of a non-segmented negative-strand RNA virus, at least one copy of a nucleic acid encoding a P protein of a non-segmented negative-strand RNA virus and a DNA flap. Definitions given above apply to these cells. In a particular embodiment, the expression vector comprises further at least one copy of a nucleic acid encoding a L protein of a non-segmented negative-strand RNA virus.

The invention is also directed to an expression retroviral-derived vector comprising a DNA flap as described above and at least one nucleic acid encoding a protein necessary for the rescue of a non-segmented negative-strand RNA virus. In a particular vector of the invention, the nucleic acid encodes a protein that is selected from the group consisting of a RNA polymerase, a N protein of a non-segmented negative-strand RNA virus, a P protein of a non-segmented negative-strand RNA virus and a L protein of a non-segmented negative-strand RNA virus.

The term “genome” refers to any nucleic acid molecule, whose presence into the cell is not dependent upon pressure selection i.e., whose presence into the cell is permanent and/or does not depend from environmental conditions. The term “genome” does not encompass plasmids. Primarily, the term “genome” refers to nucleic acid molecules present into the cell nucleus (nuclear genome), by opposition to nucleic acid molecules present into the cytoplasm, and encompasses for example chromosomes. In particular embodiment, the term “genome” also includes nucleic acid molecules present in particular cell compartments, such as organelles, for example mitochondria (mitochondrial genome) or chloroplasts (chloroplast genome). In a particular embodiment, the genome is from a eukaryotic cell.

A retroviral-derived vector, and particularly a lentiviral-derived vector and more particular a HIV-1-derived vector, is a viral genome that comprises the elements necessary for the retrotranscription, particularly the LTRs possibly mutated including deleted in part especially deleted in the U3 region, as illustrated below and advantageously the DNA flap. These LTR and DNA flap regions may be the only sequences of retroviral, especially lentiviral origin in the retroviral-derived expression vector. In no case, the retroviral-derived vector contains the nucleotide sequences encoding the full-length retroviral proteins. In a particular embodiment of the invention, the retroviral-derived vector comprises or consists of a DNA flap and at least one nucleic acid encoding a protein necessary for the rescue of a non-segmented negative-strand RNA virus as described herein, as well as the LTRs of the corresponding viral genome.

An expression vector of the invention comprises a DNA flap and a nucleic acid encoding a RNA polymerase or functional part thereof. Such a vector may be the plasmid HIV-1-TRIPΔU3.CMV-T7 deposited with the CNCM on Dec. 14, 2006, under number 1-3702, which is an HIV-1 expression vector comprising a DNA flap (TRIP), a LTR deleted in the promoter and the enhancer of the U3 domain, a CMV promoter and a nucleic acid encoding the T7 phage RNA polymerase, especially one having the sequence SEQ ID NO: 8, or the plasmid HIV-1-TRIPΔU3.CMV-nlsT7 deposited with the CNCM on Dec. 14, 2006, under number I-3703, which is an HIV-1 expression vector comprising a DNA flap (TRIP), a LTR deleted in the promoter and the enhancer of the U3 domain, a CMV promoter and a nucleic acid encoding the nuclear form of the T7 phage RNA polymerase, especially one having the sequence SEQ ID NO: 10.

An expression vector of the invention comprises a DNA flap and a nucleic acid encoding a N protein of a non-segmented negative-strand RNA virus. Such a vector may be the plasmid HIV-1-TRIPΔU3.CMV-N deposited with the CNCM on Dec. 14, 2006, under number I-3700, which is an HIV-1 expression vector comprising a DNA flap (TRIP), a LTR deleted in the promoter and the enhancer of the U3 domain, a CMV promoter and a nucleic acid encoding the N protein of the MV Schwarz, especially one having the sequence SEQ ID NO: 12.

An expression vector of the invention comprises a DNA flap and a nucleic acid encoding a P protein of a non-segmented negative-strand RNA virus. Such a vector may be the plasmid HIV-1-TRIPΔU3.CMV-P deposited with the CNCM on Dec. 14, 2006, under number 1-3701, which is an HIV-1 expression vector comprising a DNA flap (TRIP), a LTR deleted in the promoter and the enhancer of the U3 domain, a CMV promoter and a nucleic acid encoding the P protein of the MV Schwarz, especially one having the sequence SEQ ID NO: 14.

Another expression vector of the invention comprises a nucleic acid encoding an L protein of a non-segmented negative-strand RNA virus. Such a vector may be the pEMC-LSchw plasmid, deposited with the CNCM on Dec. 18, 2007, under number 1-3881. One particular nucleic acid encoding an L protein is the one having SEQ ID NO:19.

The vectors CNCM 1-3700 to 3703 quoted above all are contained in E. coli (JM109) strain, cultivated in LB medium supplemented with ampicilin (100 μg/ml) at 37° C. with shaking.

The invention relates to each and any nucleotide fragment contained in the polynucleotides inserted in the deposited plasmids referred to herein, and especially to each and any region suitable to design the insert, according to the present disclosure. It relates also to the use of these fragments for the construction of plasmids of the invention.

The four plasmids above are examples of vectors that can be used in the recombination of cells to obtain recombinant cells of the invention. However, these examples do not constitute limitations of the invention; therefore, and as described above, the N and P proteins (or their functional derivatives) can be derived from any non-segmented negative-strand RNA virus, the T7 polymerase can be any RNA polymerase, the CMV promoter can be any promoter, the TRIP DNA flap can be any DNA flap and the HIV-1 expression vector can be any vector and particularly any viral vector.

Other expression vectors of the invention comprise a DNA flap and a nucleic acid encoding a L protein of a non-segmented negative-strand RNA virus, or comprise a DNA flap and nucleic acid(s) encoding a RNA polymerase, a N protein of a non-segmented negative-strand RNA virus, a P protein of a non-segmented negative-strand RNA virus and optionally a L protein of a non-segmented negative-strand RNA virus.

The term “expression vector” indicates that, besides the elements explicitly mentioned, the vector comprises all the elements necessary to drive the expression of the nucleic acid(s) encoding the proteins of interests (expression regulatory elements), and particularly transcription regulatory elements. “Transcription regulatory element” defines any DNA regions involved in the regulation of transcription of the nucleic acid(s) integrated in the genome, and encompasses a promoter, such as CMV, EF1alpha or mPGK (murine phosphoglycerate kinase) or more generally any promoter suitable for insertion in a retroviral, especially lentiviral vector, enhancer or cis-acting regulatory elements. These elements and particularly the promoter are chosen depending upon the nature of the recombinant cells. The determination of the suitable promoter, according to the expression level sought or to the recombined cell, makes part of the knowledge of the person skilled in the art. It is noteworthy that, when the recombinant cell contains several heterologous nucleic acids (also designated polynucleotides) encoding the proteins of interest, said transcription regulatory element(s) may be unique for all the nucleic acids or shared by some of them or in contrast each nucleic acid may be associated with a particular transcription regulatory element. In the latter case, the several transcription regulatory elements may be similar or different.

The presence of the DNA flap, in all the vectors used in the recombination step, leads to the formation of a DNA triplex structure (three stranded) at the DNA flap position (the triplex structure consisting of the region between the cPPT and the CTS domains including the CTS domain), enabling the import of the nucleic acid bearing the DNA flap into the nucleus of the cell (throughout nucleus membrane pore) and further the integration into the genome of this cell. The DNA flap acts as a cis-determinant of the vector nuclear import. In a first aspect, the presence of the DNA flap is of great interest for the recombination and the integration of nucleic acid(s) into non-dividing cells, since in the absence of cell division (and membrane disintegration), the import (and thus integration of nucleic acids into the cell genome) is only identified as a residual activity; therefore, the vectors containing the DNA flap are non-replicative retroviral vectors able to transduce non-dividing cells. In a second aspect, the presence of the DNA flap is also of great interest for the recombination and the integration of nucleic acid into dividing cells, by considerably improving the percentage of cells in which the nucleic acid containing the DNA flap is integrated. The insertion of the DNA flap sequence in an expression vector, as described in the present specification, strongly increases gene transfer in vitro and in vivo by stimulating nuclear import of vector DNA (Sirven et al, 2001; Zennou et al, 2001). HIV vectors including the DNA flap sequence (TRIP vectors) are able to transduce primary B and T cells, macrophages, dendritic cells, etc with a tenfold higher efficiency than other HIV vectors that lack the DNA flap. A transduction of 80-90% of cells can be routinely obtained.

Following the recombination by the vector(s) containing a DNA flap and nucleic acid(s) encoding the at least three proteins of interest and the integration of these nucleic acids in the genome, the recombinant cells stably produce the RNA polymerase, the N protein and the P protein.

The expression vectors of the invention, used to obtain the recombinant cells of the present invention, are viral vectors, and particularly viral expression vector, such as retroviral-derived, especially lentiviral-derived vectors such as HIV-, FIV- or SIV-derived vectors. More particularly, the lentiviral-derived vector is a human lentiviral-derived vector such as an HIV expression vector, particularly HIV-1 or HIV-2-derived vector. In a preferred embodiment, the viral vector is a HIV expression vector comprising a DNA flap as described above and at least one nucleic acid encoding the at least three proteins of interest. HIV vectors are classical replacement retroviral vectors in which substantially the entire coding viral sequences are replaced by the sequence to be transferred. HIV vectors express therefore only the heterologous nucleic acid(s) contained between the two HIV LTRs or mutated LTRs and under the control of the DNA flap. These vectors can thus accommodate large polynucleotides having up to 5-6 kb. A particular embodiment of the invention is a HIV expression virus as described above, and most particularly a HIV-1 expression vector, wherein a HIV-1 LTR is deleted for the promoter and the enhancer of the U3 domain (ΔU3). This particular deletion has been previously shown to increase the expression of the nucleic acid(s) contained in the vector, and particularly when associated with a promoter.

In a particular embodiment, the recombinant cell of the invention is obtainable by recombination of its genome either with plasmids HIV-1-TRIPΔU3.CMV-T7, HIV-1-TR1PAU3.CMV-N and HIV-1-TRIPΔU3.CMV-P, or with plasmids HIV-1-TRIPΔU3.CMV-nlsT7, HIV-1-TRIPΔU3.CMV-N and HIV-1-TRIPΔU3.CMV-P.

Cells of the invention can be prokaryotic or eukaryotic cells, particularly animal or plant cells, and more particularly mammalian cells such as human cells or non-human mammalian cells. In a particular embodiment, cells, before recombination of its genome, are isolated from either a primary culture or a cell line. Cells of the invention may be dividing or non-dividing cells. As an example of cells that can be recombined to provide the recombinant cells of the invention are HEK 293 (human embryonic kidney) cells, which cell line 293 is deposited with the ATCC under No. CRL-1573. In a particular embodiment, human cells are not germinal cells and/or embryonic stem cells.

Recombinant cells of the invention can be the 293-T7-NP cell line deposited with the CNCM (Paris, France) on Jun. 14, 2006, under number 1-3618 i.e., HEK-293 cells recombined with the plasmids HIV-1-TRIPΔU3.CMV-T7, HIV-1-TRIPΔU3.CMV-N and HIV-1-TRIPΔU3.CMV-P. Another example of recombinant cells of the invention are the 293-nlsT7-NP MV cell line deposited with the CNCM on Aug. 4, 2006, under number 1-3662 i.e., HEK-293 cells recombined with plasmids HIV-1-TRIPΔU3.CMV-nlsT7, HIV-1-TRIPΔU3.CMV-N and HIV-1-TRIPΔU3.CMV-P.

In a further embodiment of the invention, the recombinant cells of the invention are further recombined by an expression vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus. The expression of the L protein may be temporary and driven by a plasmid not containing DNA flap, or in contrast be stable and driven by a vector containing a DNA flap as defined above. The recombination by a plasmid or vector bearing the at least one copy of the nucleic acid encoding the L protein may be simultaneous to or subsequent to the recombination by the vector(s) containing the coding sequence(s) of the RNA polymerase, the N protein and the P protein.

Therefore, the present invention also refers to a cell stably producing a RNA polymerase, a N protein of a non-segmented negative-strand RNA virus and a P protein of a non-segmented negative-strand RNA virus, or functional derivatives thereof, and producing, stably or not, a L protein of a non-segmented negative-strand RNA virus.

The L protein is derived from any non-segmented negative-strand RNA virus quoted in Table 1. In a particular embodiment, the L protein is from the same non-segmented negative-strand RNA virus as the N protein and/or the P protein, and particularly from the same virus strain. In another embodiment, the L protein is from a different non-segmented negative-strand RNA virus than the N protein and/or the P protein.

In particular embodiment, the L protein is from a Paramyxoviridae virus, preferably a Paramyxovirinae virus, and most preferably a Morbillivirus virus. As an example of Morbillivirus is the Measles virus (MV), in particular an attenuated non immunosuppressive strain, e.g. an approved strain for a vaccine, and especially the Schwarz MV strain, or even the Edmonston (Ed) strain. A particular L protein is the one of the MV virus (SEQ ID NO: 16) or the one encoded by the sequence inserted in pEMC-LSchw plasmid and especially the sequence found between nucleotides 1425 and 7976 of SEQ ID NO:19.

In a particular embodiment, the sequence of the L protein must not be modified with respect to the wild type L protein and must be functional i.e., enabling the production of particles or virus when transcomplemented with N and P proteins and a T7 polymerase in a host cell. A test to determine the effective functionality of a clone bearing the L protein is carried out by transfecting a competent cell with vector(s) encoding the N protein, the P protein and the T7 (or nlsT7) polymerase, a vector encoding the L protein to be tested, and a minigenome comprising a leader, a promoter, a reporter gene (such a GFP) and a trailer. The functionality of the L clone is revealed by the production of particles expressing the reporter gene.

The present invention also describes a cell according to the present specification further recombined with a non-segmented negative-strand cDNA clone of a non-segmented negative strand RNA virus i.e., the antigenomic RNA (+) strand of the virus genome. “cDNA” used for the description of the nucleotide sequence of the molecule of the invention merely relates to the fact that originally said molecule is obtained by reverse transcription of the genomic (−) RNA genome of viral particles of a non-segmented negative strand RNA virus, particularly of the measles virus, and most preferably the full-length genomic (−) RNA genome of viral particles of a non-segmented negative strand RNA virus. This should not be regarded as a limitation for the methods used for the preparation of this cDNA clone. The invention thus encompasses, within the expression “cDNA”, every DNA provided it has the above defined nucleotide sequence. Purified nucleic acids, including DNA, or plasmids are thus encompassed within the meaning cDNA according to the invention, provided said nucleic acid, especially DNA fulfils the above-given definitions.

In a particular embodiment, the cDNA clone of a non-segmented negative strand RNA virus contains, upstream of the viral sequences, transcription regulatory elements. In a preferred embodiment, these elements are the same as the one(s) located in the expression vector(s) comprising the N, P and/or L proteins described above. In a more preferred embodiment, the element is a T7 RNA polymerase promoter.

In an embodiment, the cDNA clone of a non-segmented negative-strand RNA virus is from the same non-segmented negative-strand RNA virus as the N protein and/or the P protein and/or the L protein, and particularly from the same virus strain. In another embodiment, the cDNA clone of a non-segmented negative strand RNA virus is from a different non-segmented negative-strand RNA virus than the N protein and/or the P protein and/or the L protein.

In particular embodiment, the cDNA clone is from a non-segmented negative strand RNA virus, such as a Paramyxoviridae virus, preferably a Paramyxovirinae virus, and most preferably a Morbillivirus virus. As an example of Morbillivirus is the Measles virus (MV), in particular an attenuated non immunosuppressive strain, e.g. an approved strain for a vaccine, and especially the Schwarz MV strain or the Edmonston (Ed) strain. Moreover, the nucleotide sequence of the non-segmented negative-strand cDNA clone may be modified as compared to the wild type strain or virus, such a defined below.

The invention also concerns cultures of cells wherein said cells are those defined throughout the specification, and particularly cultures of cells stably producing a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a phosphoprotein (P) of a non-segmented negative-strand RNA virus, or functional derivatives thereof. In another embodiment, the invention also concerns cultures of cells stably producing a RNA polymerase, a N protein of a non-segmented negative-strand RNA virus and a P protein of a non-segmented negative-strand RNA virus, or functional derivatives thereof, and producing, stably or transitory, a L protein of a non-segmented negative-strand RNA virus or functional derivatives thereof.

In an embodiment, the cells culture to be recombined is a primary culture i.e., a culture prepared from cells or tissues directly obtained from an animal (optionally non-human) or a plant. In another embodiment, the cells culture to be recombined is a cell line i.e., a population of cells resulting from the first subculture of a primary culture or from subsequent serial passaging of the cells.

In another aspect, the present invention also relates to various methods to produce infectious, recombinant, non-segmented negative-strand virus, using the cells of the invention.

A first method to produce infectious, recombinant, non-segmented negative-strand virus comprises or consists in:

-   -   a. recombining a cell or a culture of cells stably producing a         RNA polymerase, a nucleoprotein (N) of a non-segmented         negative-strand RNA virus and a polymerase cofactor         phosphoprotein (P) of a non-segmented negative-strand RNA virus,         with cDNA clone of a non-segmented negative strand RNA virus and         with a vector comprising a nucleic acid encoding a RNA         polymerase large protein (L) of a non-segmented negative-strand         RNA virus,     -   b. transferring said recombinant cell or culture of recombinant         cells onto cells competent to sustain the replication and         production of non-segmented negative-strand RNA virus, and     -   c. recovering the infectious, recombinant, non-segmented         negative-strand RNA virus from the co-culture of step b.

A second method according to the invention is a method to produce infectious, recombinant, non-segmented negative-strand RNA virus comprising or consisting of:

-   -   a. recombining a cell or a culture of cells stably producing a         RNA polymerase, the nucleoprotein (N) of a non-segmented         negative-strand RNA virus and the polymerase cofactor         phosphoprotein (P) of a non-segmented negative-strand RNA virus,         with a cDNA clone of a non-segmented negative strand RNA virus         and with a vector comprising a nucleic acid encoding a RNA         polymerase large protein (L) of a non-segmented negative-strand         RNA virus, and     -   b. recovering the infectious, recombinant, non-segmented         negative-strand RNA virus from said recombinant cell or culture         of recombinant cells.

As used herein, “recombining” means introducing at least one polynucleotide into a cell, for example under the form of a vector, said polynucleotide integrating (entirely or partially) or not integrating into the cell genome (such as defined above). According to a particular embodiment recombination can be obtained with a first polynucleotide which is a cDNA clone of a non-segmented negative strand RNA virus, whose definition, nature and optional modifications are discussed elsewhere in the present specification. Recombination can, also or alternatively, encompasses introducing a polynucleotide which is a vector encoding a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus, whose definition, nature and stability of expression has been described herein.

In these methods, the cell or a culture of cells stably producing a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a polymerase cofactor phosphoprotein (P) of a non-segmented negative-strand RNA virus is a cell as defined in the present specification or a culture of cells as defined in the present specification, i.e., are also recombinant cells to the extent that they have been modified by the introduction of one or more polynucleotides as defined above. In a particular embodiment of the invention, the cell or culture of cells, stably producing the RNA polymerase, the N and P proteins, does not produce the L protein of a non-segmented negative-strand RNA virus or does not stably produce the L protein of a non-segmented negative-strand RNA virus, e.g., enabling its transitory expression or production.

“Transfer” as used herein refers to the plating of the recombinant cells onto a different type of cells, and particularly onto monolayers of a different type of cells. These latter cells are competent to sustain both the replication and the production of infectious, recombinant, non-segmented negative-strand RNA viruses i.e., respectively the formation of infectious viruses inside the cell and possibly the release of these infectious viruses outside of the cells. This transfer results in the co-culture of the recombinant cells of the invention with competent cells as defined in the previous sentence. The above transfer may be an additional, i.e., optional, step when the recombinant cells are not efficient virus-producing culture i.e., that infectious viruses can not be efficiently recovered from these recombinant cells. This step is introduced after further recombination of the recombinant cells of the invention with a cDNA clone of a non-segmented negative-strand RNA virus, and optionally a vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus.

In a particular embodiment of the invention, a transfer step is required since the recombinant cells, usually chosen for their capacity to be easily recombined are not efficient enough in the sustaining and production of recombinant infectious viruses. In said embodiment, the cell or culture of cells of step a. of the above-defined methods is a recombinant cell or culture of recombinant cells according to the invention, particularly recombinant HEK-293 cells such as the 293-T7-NP cell line deposited with the CNCM on Jun. 14, 2006, under number 1-3618 or 293-nlsT7-NP MV cell line deposited with the CNCM on Aug. 4, 2006, under number I-3662.

Cells competent to sustain the replication and production of non-segmented negative-strand RNA virus may be any cell type that can be co-cultivated with the recombinant cells of the invention but not necessarily cells of the same Kingdom, Phylum, Class, Order, Family, Genus or Species. Examples of competent cells are Vero (African green monkey kidney) cells or CEF (chick embryo fibroblast) cells. CEF cells can be prepared from fertilized chicken eggs as obtained from EARL Morizeau (8 rue Moulin, 28190 Dangers, France), from any other producer of fertilized chicken eggs or from MRC5 cells (ATCC CCL171; lung fibroblast).

In another embodiment of the invention, the transfer step is not needed and thus not carried out. This is one of the advantages of the present invention to provide a method to produce infectious, recombinant, non-segmented negative-strand RNA viruses that is easy to carry out, faster and cheaper than the conventional methods and enabling the recovery of recombinant infectious viruses free of contaminants. This can be achieved with the recombinant cells of the invention that have the features of:

-   -   stably producing a RNA polymerase, a nucleoprotein (N) of a         non-segmented negative-strand RNA virus and a phosphoprotein (P)         of a non-segmented negative-strand RNA virus, and     -   from which infectious recombinant viruses can be efficiently         recovered, without contaminations by unwanted viruses and/or         other type of cells.

The “Recovery of infectious recombinant virus” as used herein refers to any means by which the infectious viruses, previously produced by the cells, are released from the cells, and isolated from the cultured cells. The recovery is said to be “direct” when the infectious recombinant viruses are recovered from recombinant cells of the invention, without involvement of other cell type(s). In contrast, the recovery is said to be “indirect” when the infectious recombinant viruses are recovered via another cell type than the recombinant cells of the invention. As mentioned earlier, the present invention is the first to report the direct recovery of infectious, recombinant, non-segmented negative-strand RNA virus.

In particular methods of the invention, the recombining step does not comprise the steps of recombining a cell or a culture of cells stably producing a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a polymerase cofactor phosphoprotein (P) of a non-segmented negative-strand RNA virus, with a vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus. In that case, the recombinant cells of the invention have been selected for their capacity to express the L protein and especially have been previously recombined with a vector comprising a nucleic acid encoding a RNA polymerase large protein (L), the nucleic acid encoding the L protein being integrated in the cell genome or not.

When appropriate vectors bearing accessory proteins (non-P, non-L or non-N proteins, or non-RNA polymerase) may optionally be used in the methods of the invention, particularly when a genome or a cDNA clone, deleted for these proteins, is used. Such accessory proteins are the C protein, the V protein, the NS1 protein, the NS2 protein, the M protein, the M2 protein and/or the SH proteins. The vector(s) bearing the coding sequences of these accessory proteins may optionally comprises a cDNA flap as defined above.

The stability of the production of the RNA polymerase, N protein and P protein in the recombinant cells of the invention leads to some advantages according to the methods previously described in the art:

-   -   the method of the invention does not necessarily comprise a         transfer step;     -   the method does not comprise heat shock step as reported in         Parks et al. (1999). Indeed, this step has been shown to improve         the efficiency of the synthesis of viral N and P proteins, as         well as RNA polymerase, which proteins are synthesized from         nucleic acids borne on plasmids. In the present invention,         however, the nucleic acids are integrated into the cell genome,         and the expression of these proteins has been shown to be         stable, and/or at a level appropriate to initiate the de novo         encapsidation.     -   the method produces large quantities of infectious viruses,         since the production of the RNA polymerase, N protein and P         protein is stable, and not dependent on their expression from         plasmids. Therefore, about 100-400 out of 10⁶ recombined cells         transmit infectious viruses after recombination (number of         rescue events). This is mostly superior to the 1-6 out of 10⁶         transfected cells obtained with the method of Radecke et al.         (1995). In a particular embodiment of the method, the number of         rescue events, for 10⁶ recombined cells, is more than 20, more         than 50, more than 100, more than 200, more than 300, more than         400 or more than 500.

Finally, another advantage of the invention is the large variety of cells that can be recombined, and used to perform the invention. Indeed, the recombinant cells can be any eukaryotic cell, particularly any mammalian cell, either non-human cell or human cell. In a particular embodiment, the recombinant cells of the invention are human fibroblasts, especially MRC5 cell line (human lung fibroblasts). The invention is particularly useful for cells that do not divide.

According to the invention, the cDNA clone of a non-segmented negative-strand of a RNA virus is from a MV virus, particularly an attenuated virus in particular an attenuated non immunosuppressive strain, e.g. an approved strain for a vaccine, such as the Schwarz MV strain. A cDNA clone is a DNA sequence encoding the full-length antigenome of a non-segmented negative-strand RNA virus.

In a particular embodiment of the invention, the N, P and L proteins as well as the cDNA clone are from the same virus, that can be any virus of Table I, particularly a MV virus as disclosed above such as the Schwarz MV strain of measles virus. The nucleotide sequences of the Edmonston B. strain and of the Schwarz strain have been disclosed in WO 98/13505. Independently of the nature of the N, P and L proteins and the cDNA clone of the non-segmented negative-strand RNA virus, the RNA polymerase is the T7 RNA polymerase. A particular cDNA sequence is the sequence of the cDNA of the Schwarz strain as defined in SEQ ID NO: 18. Such a cDNA can be obtained from pTM-MVSchw, that is a plasmid derived from Bluescript containing the complete sequence of the measles virus, vaccine strain Schwarz, under the control of the promoter of the T7 RNA polymerase. Its size is 18967 nt.

Alternatively, the cDNA clone of a non-segmented negative strand RNA virus is derived from any virus of Table I. A particular recombinant measles virus from which the cDNA clone is derived from is the Schwarz strain and especially an approved vaccine Schwarz strain such as that produced under the trademark Rouvax, available from Aventis Pasteur (France).

An “attenuated strain” is defined herein as a strain that is avirulent or less virulent than the parent strain in the same host, while maintaining immunogenicity and possibly adjuvanticity when administered in a host i.e., preserving immunodominant T and B cell epitopes and possibly the adjuvanticity such as the induction of T cell costimulatory proteins or the cytokine IL-12. In a particular embodiment, the attenuated strain is an “approved vaccine strain” i.e., a strain certified for use in vaccine production by one national or regional health authority having granted a marketing approval for this product (legal designation). Accordingly, an “approved vaccine strain” has been shown to be safe, stable and able to provide effective protection (immunogenicity and adjuvanticity). Stability of a strain is measured by assessing that the properties of the strain remain substantially unchanged after numerous passages on the same certified cell line.

“Derived from” as used herein means any cDNA clone whose nucleotide sequence is modified as compared to the one of the wild type virus or strain. This modification may be at least one substitution, deletion or insertion in the nucleotide sequence and particularly in the coding sequence of a protein of the virus or strain. In another embodiment, the nucleotide sequence is modified by the insertion of at least one heterologous nucleic acid(s) i.e., a sequence that is not naturally present in the virus or the strain in which the at least one nucleic acid(s) is inserted or a sequence which is not derived from the antigens of measles viruses. Moreover, the cDNA clone may be modified by deletion of part(s) of the wild-type viral genome, and insertion of heterologous nucleic acids.

In a preferred embodiment, it is pointed out that the derived cDNA clone, consisting or comprising one or several heterologous nucleic acid(s), meets the so-called rule of 6. Therefore, the derived cDNA clone is a polyhexameric length, i.e., is a multiple of six. This requirement is especially achieved for cDNA clones derived from Paramyxoviridae, and in particular measles viruses. Some non-segmented negative-strand RNA viruses do not comply with this rule, as known from the skilled person in the art.

Any heterologous nucleic acid can be inserted in the nucleotide sequence of the cDNA clone, as far as the insertion does not prevent the production of infectious recombinant non-segmented negative-strand virus (permissive sites). In a particular embodiment, the insertion or deletion of the native viral genome provides a polynucleotide which is a multiple of six. Therefore, even though the genome length is not a multiple of six, the modification consists of six or multiple of six deletions and/or insertions.

Therefore, the heterologous nucleic acid sequences may encode one or several peptides able to elicit a humoral and/or cellular immune response (such as CTL or CD4 response) in a determined host, against the organism or organisms especially the pathogenic organism(s), for example the virus, especially retrovirus, flavivirus or coronavirus, of the bacterium or parasites from which it (they) originate(s). Accordingly, the amino acid sequence of such peptide is one which comprises at least one epitope of an antigen, especially a conserved epitope, which epitope is exposed naturally on the antigen or is obtained or exposed as a result of a mutation or modification or combination of antigens. Heterologous nucleic acids, which can be inserted in the cDNA clones, encode especially structural antigens (including antigenic fragments thereof or derivatives of said antigens or fragments) of viruses including retroviruses such as human retroviruses especially lentivirus, in particular HIV-1 or HIV-2, flavivirus or coronavirus envelope, such as envelop or capsid antigen. Particularly, such antigens are especially from envelopes of AIDS viruses including HIV-1 or HIV-2, from capsid of HIV or from envelopes of the Yellow Fever Virus or envelopes from the West Nile Virus, or from envelopes of the Dengue virus (DV), envelopes of the Japanese encephalitis virus (JEV) or envelope of the SARS-associated coronavirus. Other retroviral, flaviviral or coronavirus antigens may however be advantageously used in order to derive recombinant measles viruses capable of eliciting antibodies against said retroviruses or flaviviruses, and/or capable of eliciting the production of neutralizing antibodies against the retrovirus or flaviviruses. In another embodiment, the peptide encoded or encompassed by the heterologous nucleic acid sequences is tumoral antigen or an antigen specifically expressed on the cell surface of cancer cells. According to another embodiment of the invention, the sequences encode multiepitopes or antigens that alternatively or additionally also elicit a cellular immune response against the retrovirus or flaviviruses.

Advantageously, the recombinant measles viruses produced by the method of the invention may also elicit a humoral and/or cellular immune response against measles virus. This response is however not mandatory provided the immune response against the epitope, or multiepitopes or antigens disclosed above is indeed obtained.

In a preferred embodiment of the invention, the heterologous nucleic acid encodes a protein from an HIV retrovirus, particularly an envelope antigen of HIV and especially a peptide derived from an envelope protein or glycoprotein of HIV-1 or HIV-2. The antigens of interest in this respect are especially gp160, gp120 and gp41 of HIV-1 or gp140, GAG or TAT of HIV-1. In a particular embodiment of the invention, the heterologous amino acid sequence is derived from a recombinant gp160, gp120 of HIV-1 or gp140, GAG or TAT of HIV-1.

In another embodiment, the V1, V2 and/or V3 loops of the gp120 (or gp160) antigen are deleted or deleted in part, individually or in combination in such a way that conserved epitopes are exposed on the obtained recombinant gp120 antigen. The V1, V2 and V3 loops of the gp120 (or gp160) antigen of HIV-1 have been especially disclosed in Fields virology (Fields B. N. et al. Lippincott Raven publishers 1996, p. 1953-1977).

In another embodiment, the heterologous nucleic acid encodes a peptide that is derived from the gp120 (or gp160) antigen of HIV-1, wherein the V1, V2 and/or V3 loops of the gp120 (or gp160) antigen are substituted or substituted in part, individually or in combination, in such a way that conserved epitopes are exposed on the obtained recombinant gp120 (or gp160) antigen.

In another embodiment, the heterologous nucleic acid encodes a peptide that is derived from an envelope antigen of HIV-1 especially is derived from the gp120 antigen in such a way that the V1 and V2 loops are deleted and the V3 loop is substituted for the sequence AAELDKWASAA.

In another embodiment, the heterologous nucleic acid encodes a peptide that is gp160ΔV3, gp160ΔV1V2, gp160ΔV1V2V3, gp140ΔV3, gp140ΔV1V2, gp140ΔV1V2V3.

Preferred cDNA clones containing epitopes from HIV, WNV, YFV, DV or JEV are vectors defined in Table II deposited at the CNCM (Collection Nationale de Culture de Microorganismes—Institut Pasteur—Paris, France), and whose features are given below.

TABLE II Strain from which the sequence is derived from Vector name Deposit number Date of deposit Edmonston B. strain pMV2(EdB)gp160[delta]V3HIV89.6P CNCM I-2883 Jun. 12, 2002 pMV2(EdB)gp160HIV89.6P CNCM I-2884 pMV2(EdB)gp140HIV89.6P CNCM I-2885 pMV3(EdB)gp140[delta]V3HIV89.6P CNCM I-2886 pMV2(EdB)-NS1YFV17D CNCM I-2887 pMV2(EdB)-EnvYFV17D CNCM I-2888 Schwarz strain pTM-MVSchw2-Es(WNV) CNCM I-3033 May 26, 2003 pTM-MVSchw2-GFPbis CNCM I-3034 pTM-MVSchw2-p17p24[delta]myr(HIVB) CNCM I-3035 pTM-MVSchw3-Tat(HIV89-6p) CNCM I-3036 pTM-MVSchw3-GFP CNCM I-3037 pTM-MVSchw2-Es (YFV) CNCM I-3038 pTM-MVSchw2-gp140 [delta] V1 V2 V3 (HIV89-6) CNCM I-3054 Jun. 19, 2003 pTM-MVSchw2-gp140 [delta] V3 (HIV89-6) CNCM I-3055 pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6) CNCM I-3056 pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6) CNCM I-3057 pTM-MVSchw2-Gag SIV239 p17-p24 [delta] myr-3-gp140 (HIV89-6) CNCM I-3058 pTM-MVSchw2 [EDIII + M¹⁻⁴⁰]WNV (IS-98-ST1) CNCM I-3440 May 26, 2005 pTM-MVSchw2 [EDIII + apoptoM]DV1 (FGA89) CNCM I-3442 pTM-MVSchw2 [EDIII] JEV (Nakayama) CNCM I-3441

I-2883 (pMV2(EdB)gp160[delta]V3HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp160ΔV3+ELDKWAS of the virus SVIH strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21264 nt.

I-2884 (pMV2(EdB)gp160HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp160 of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21658 nt.

I-2885 (pMV2(EdB)gp140HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp140 of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21094 nt.

I-2886 (pMV3(EdB)gp140[delta]V3HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp140ΔV3(ELDKWAS) of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21058 nt.

I-2887 (pMV2(EdB)-NS1YFV17D) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the NS1 gene of the Yellow Fever virus (YFV 17D) inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 20163 nt.

I-2888 (pMV2(EdB)-EnvYFV17D) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the Env gene of the Yellow Fever virus (YFV 17D) inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 20505 nucleotides.

I-3033 (pTM-MVSchw2-Es(WNV)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the secreted envelope, (E) of the West Nile virus (WNV), inserted in an ATU.

I-3034 (pTM-MVSchw2-GFPbis) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the GFP inserted in an ATU.

I-3035 (pTM-MVSchw2-p17p24[delta]myr(HIVB)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the gag gene encoding p17p24Amyrproteins of the HIVB virus inserted in an ATU.

I-3036 (pTMVSchw3-Tat(HIV89-6p)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the Tat gene of the virus strain 89.6P inserted in an ATU.

I-3037 (pTM-MVSchw3-GFP) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) under the control of the T7 RNA polymerase promoter and expressing the gene of the GFP gene inserted in an ATU having a deletion of one nucleotide.

I-3038 (pTM-MVSchw2-Es) (YFV) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) under the control of the T7 RNA polymerase promoter and expressing the gene of the secreted protein of the Fever virus (YFV) inserted in an ATU.

I-3054 (pTM-MVSchw2-gp140 [delta] V1 V2 V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp140 [delta] V1 V2 (HIV 89-6) inserted in an ATU.

I-3055 (pTM-MVSchw2-gp140 [delta] V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp14 [delta] V3 (HIV 89-6) inserted in an ATU.

I-3056 (pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp160 [delta] V1 V2 V3 (HIV 89-6) inserted in an ATU.

I-3057 (pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp160 [delta] V1 V2 (HIV 89-6) inserted in an ATU.

I-3058 (pTM-MVSchw2-Gag SIV239 p17-p24 [delta] myr-3-gp140 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding Gag SIV239 p17-p24 [delta] myr-3-gp140 (HIV89-6) inserted in an ATU.

I-3440 (pTM-MvSchw2-[EDIII+M¹⁻⁴⁰] WNV (IS-98-ST1)) is a plasmid derived from PTM containing the cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) and an additional expression unit located between the P and M genes, this unit containing the nucleotide sequence of the domain III from the envelop protein of the West Nile virus (WNV) (WNV IS-98-ST1) fused to the sequence 1-40 of the membrane protein M.

I-3442 (pTM-MvSchw2-[EDIII+ApoptoM] DV1 (FGA89)) is a plasmid derived from PTM containing the cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) and an additional expression unit located between the P and M genes, this unit containing the nucleotide sequence of the domain III from the envelop protein of dengue-1 virus (strain FGA89) fused to the apoptotic sequence of the membrane protein M.

I-3441 (pTM-MvSchw2-[EDIII] JEV (Nakayama)) is a plasmid derived from PTM containing the cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) and an additional expression unit located between the P and M genes, this unit containing the nucleotide sequence of the domain III from the envelop protein of the Japanese encephalitis virus (JEV), strain Nakayama.

In a particular embodiment, the heterologous nucleic acid encodes a peptide that is derived from an antigen of the Yellow Fever virus selected among the envelope (Env), the NS1 proteins or immunogenic mutants thereof. When the heterologous DNA sequence present in the recombinant measles virus vector of the invention is derived from the Yellow Fever Virus (YFV), it is advantageously selected among YFV 17D 204 commercialized by Aventis Pasteur under the trademark Stamaril®.

In another particular embodiment, the heterologous nucleic acid encodes a peptide that is derived from an antigen of the West Nile virus selected among the envelope (E), premembrane (preM) or immunogenic mutants thereof. When the heterologous DNA sequence present in the recombinant measles virus vector of the invention is derived from the West Nile Virus (WNV), it is advantageously selected among the neurovirulent strain IS 98-ST1.

The heterogeneous nucleic acid may encode a tumour-specific antigen (TSA) or a tumour-associated antigen (TAA).

Another advantage of the invention is the possibility to insert in the cDNA clone of a non-segmented negative-strand RNA virus, long heterologous nucleic acid or a large number of heterologous nucleic acids. Therefore, the cDNA clone may be modified by insertion of one or several heterologous nucleic acids whose total sequence is at least 5 kb.

The invention relates to each and any nucleotide fragment contained in the polynucleotides inserted in the deposited plasmids referred to herein, and especially to each and any region suitable to design the insert, according to the present disclosure. It relates also to the use of these fragments for the construction of plasmids of the invention.

The invention also relates to methods to produce recombinant cells stably expressing the three or at least the three following proteins, a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a phosphoprotein (P) of a non-segmented negative-strand RNA virus, or functional derivatives thereof, comprising or consisting in either:

-   -   a. recombining a cell with at least:         -   an expression vector comprising a DNA flap, and at least one             copy of a nucleic acid encoding a RNA polymerase,         -   an expression vector comprising a DNA flap, and at least one             copy of a nucleic acid encoding a N protein of a             non-segmented negative-strand RNA virus, and         -   an expression vector comprising a DNA flap, and at least one             copy of a nucleic acid encoding a P protein of a             non-segmented negative-strand RNA virus, and     -   b. selecting the cells that stably produce at least a RNA         polymerase, a nucleoprotein (N) of a non-segmented         negative-strand RNA virus and a phosphoprotein (P) of a         non-segmented negative-strand RNA virus, or functional         derivatives thereof.         or     -   a. recombining a cell with at least an expression vector         comprising:         -   at least one copy of a nucleic acid encoding a RNA             polymerase under the control of a promoter,         -   at least one copy of a nucleic acid encoding a N protein of             a non-segmented negative-strand RNA virus under the control             of a promoter,         -   at least one copy of a nucleic acid encoding a P protein of             a non-segmented negative-strand RNA virus under the control             of a promoter, and         -   a DNA flap, and     -   b. selecting the cells that stably produce at least a RNA         polymerase, a nucleoprotein (N) of a non-segmented         negative-strand RNA virus and a phosphoprotein (P) of a         non-segmented negative-strand RNA virus, or functional         derivatives thereof.

In a particular embodiment of the invention, the method to produce recombinant cells comprises further recombining the recombinant cells obtained in the a. step of the method above with an expression vector comprising a nucleic acid encoding a RNA polymerase large protein (L) or a functional derivative thereof of a non-segmented negative-strand RNA virus and selecting the cells that stably produce at least a RNA polymerase, a nucleoprotein (N) of a non-segmented negative-strand RNA virus and a phosphoprotein (P) of a non-segmented negative-strand RNA virus, and that produce a large protein (L) of a non-segmented negative-strand RNA virus or functional derivatives thereof.

The present invention is also directed to the use of recombinant cells of the invention, as described in the present specification, as helper cells, especially as helper cells in the production of infectious, recombinant, non-segmented negative-strand RNA virus.

Further embodiments and characteristics of the invention defined are found in the following examples and figures.

EXAMPLES

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Cells and Viruses

Vero (African green monkey kidney) cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). Human kidney 293 (HEK-293) cells were grown in DMEM supplemented with 10% FCS. Human diploid MRC5 cells were grown as monolayers in DMEM supplemented with 10% FCS. Chicken embryo fibroblastic cells (CEF) were prepared as follows: fertilized chicken eggs (Morizeau, Dangers, France) were incubated at 38° C. for 9 days. Embryos were collected under sterile conditions. Head, limbs and viscera were removed and embryos were chopped then trypsinized for 5-10 minutes at 37° C. (Trypsin/EDTA 2.5 g/L). After filtration (70 μm) and several washes in DMEM high glucose/10% FCS, cells were seeded (5-7 10⁶ cells per Petri dish) and incubated overnight at 37° C. before use for virus infection.

Plasmid Constructions

To allow the easy recombination of additional sequences using the Gateway® recombination system (Invitrogen), the Gateway® cassette (attb1/attb2 Seq) was introduced by ligation into the HIV-1-TRIP-ΔU3-BSX plasmid vector (Zennou et al., 2000) linearized by SmaI digestion. The T7 RNA polymerase gene was amplified from pAR-1173 plasmid (Brookhaven National Laboratory, ref) by PCR using PfuTurbo DNA polymerase (Stratagene) and the following primers containing the Gateway® recombination sequences (underlined):

AttB1-T7Pol: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATGGAATTCTCT GACATCGAACTGGCT-3′ AttB2-retourT7Pol: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTATCACGCGAACGCGAA GTCCGACTCTAAGATGTC-3′

A nuclear form of T7 RNA polymerase (nlsT7) was also amplified from pAR-3288 plasmid (Brookhaven National Laboratory, ref) using the following primers containing a nuclear localization signal (in bold) and the Gateway® recombination sequences (underlined):

AttBl-SV40nls: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATGGCACCAAAA AAGAAGAGAAAGGTA-3′ AttB2-retourT7Pol: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTATCACGCGAACGCGAA GTCCGACTCTAAGATGTC-3′

Using the same approach, the Schwarz MV N and P genes were amplified by PCR from pTM-MVSchw plasmid, which contains a full-length infectious Schwarz MV antigenome (Combredet et al., 2003). The following primers containing the Gateway® recombination sequences (underlined) were used:

AttB1-N: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCCACACTTTTA AGGAGCTTAGCA-3′ AttB2-N: 5′-GGGACCACTTTGTACAAGAAAGCTGGGTGTGTACTAGTCTAGAAGA TTTCTGTCATTGTA-3′ AttB1-P:  5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCAGAAGAGCAG GCACGCCAT-3′ AttB2-P: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTACTACTTCATTATT ATCTTCATCAGCATCTGGTGGA-3′

The different PCR fragments encoding the T7 RNA polymerase, the nlsT7 RNA polymerase and the MV N and P proteins were then introduced into the pDONR™207 entry plasmid (Invitrogen) and recombined in the modified HIV-1-TRIP-AU3-BSX plasmid using the Gateway® recombination system (Invitrogen). The different recombinant vector plasmids obtained (HIV-1-TRIP delta U3.CMV-T7, HIV-1-TRIP delta U3.CMV-nlsT7, HIV-1-TRIP delta U3.CMV-N and HIV-1-TRIP delta U3.CMV-P) were fully sequenced. These vectors were deposited with the CNCM on Dec. 14, 2006, under respectively number I-3702, I-3703, I-3700 and I-3701.

The plasmid pEMC-LSchw expressing the large polymerase (L) protein from Schwarz MV was constructed in a similar way as described in Radecke et al. (1995). The 6552 nucleotide long sequence of the Schwarz L gene was taken from pTM-MVSchw plasmid (Combredet et al., 2003) and inserted into the pEMC-La plasmid previously described in Radecke et al. (1995), using classical cloning procedures. This plasmid was deposited with the CNCM on Dec. 18, 2007 under number I-3881.

Production of Vector Particles

Vector particles have been produced by co-transfection of HEK-293 cells using calcium-phosphate procedure with either HIV-1-TRIP delta U3.CMV-T7, HIV-1-TRIP delta U3.CMV-nlsT7, HIV-1-TRIP delta U3.CMV-N, or HIV-1-TRIP delta U3.CMV-P vector plasmids, an encapsidation plasmid expressing HIV-1 gag and pol genes, and a plasmid expressing the VSV-G envelope glycoprotein (pHCMV-G) as described in (Zennou et al., 2000). The amount of Gag p24 antigen in stocks of vector particles concentrated by ultracentrifugation was determined using HIV-1 p24 ELISA (Perkin Elmer LifeSciences).

Generation of Cell Lines 293-T7-MV

Cells (HEK-293) were seeded into 35 mm wells one day before transduction by TRIP-T7 and TRIP-nlsT7 lentiviral vectors. Vectors (500 ng/ml p24) were added in DMEM supplemented with 10% FCS. During 8 days, the same amount of vector was repeatedly added every day on cells. Cells were expanded every two days. After each passage, the T7 RNA polymerase activity of the cells was determined. A 35 mm cell culture was transfected with 5 μg of pEMC-Luc using the calcium-phosphate procedure, and the luciferase activity in 1/20 of the cleared cell lysate harvested one day after transfection was measured in a luminometer. The luciferase activity increased after each additional transduction and remained maximal between the 7^(th) and the 8^(th) transduction. The absence of cytotoxicity of T7 RNA polymerase expression was demonstrated after each transduction by quantifying cell viability using the trypan blue-exclusion method and comparison to non-transduced cells. After 8 steps of transduction, two cell populations were generated with a very high T7 RNA polymerase activity, either cytoplasmic (293-T7) or nuclear (293-nlsT7).

The two cell populations (293-T7 and 293-nlsT7) were then co transduced simultaneously by TRIP-N and TRIP-P vectors. Vectors (TRIP N: 390 ng/ml p24 and TRIP P: 330 ng/ml p24) were added on cells seeded in 35 mm wells. During 10 days, the same amount of both vectors was repeatedly added every day on cells. Cells were expanded every two days, After 10 rounds of transduction, the expression of MV N and P proteins was analyzed on the total cell populations by western blotting using 1/20 of the total lyzate of a 35 mm well. The expression of both proteins was comparable to that of similar number of infected Vero cells (FIG. 2). Transduced cells were then cloned by limiting dilution. Cells were seeded in 96-well plates at a dilution of ⅓ cell per well. After 2 weeks, the first clones were selected. About 100 clones of each 293-T7-NP and 293-nlsT7-NP cells were expanded to 24-well plates, then to 35 mm wells. The expression of MV N and P proteins was analyzed on 20 clones by western blotting using 1/20 of the total lyzate of a 35 mm well. The expression of both proteins was comparable to that of similar number of infected Vero cells (FIG. 2). The T7 RNA polymerase activity was measured for each clone as described above, A number of clones with a very high luciferase activity and a similar level of MV N and P expression were selected. The clones, listed below, were amplified and frozen at −180° C. in DMEM/30% FCS/10% DMSO at a density of 10⁷ cells/ml: 293-T7-NP1, 293-T7-NP3, 293-T7-NP5, 293-T7-NP7, 293-T7-NP8, 293-T7-NP10, 293-T7-NP13, 293-T7-NP14, 293-T7-NP20, 293-T7-NP28, 293-T7-NP31, 293-T7-NP33, 293-nlsT7-NP1, 293-nlsT7-NP5, 293-nlsT7-NP6, 293-nlsT7-NP13, 293-nlsT7-NP14, 293-nlsT7-NP15, 293-nlsT7-NP30 and 293-nlsT7-NP40.

Rescue of Schwarz MV Using 293-T7-NP and 293-nls T7-NP Helper Cells

To evaluate the capacity of the different helper 293-T7-NP and 293-nlsT7-NP cell clones generated to efficiently rescue MV from cDNA, we used the plasmid pTM-MVSchw-eGFP (Combredet et al., 2003) to rescue a recombinant Schwarz MV expressing the green fluorescent protein (eGFP). We used a similar system as described previously (Radecke et al., 1995; Parks et al., 1999; Combredet et al., 2003). Helper cells 293-T7-NP or 293-nlsT7-NP were transfected using the calcium phosphate procedure with pTM-MVSchw-eGFP (5 μg) and the plasmid pEMC-LSchw expressing the Schwarz MV polymerase (L) gene (20-100 ng). After overnight incubation at 37° C., the transfection medium was replaced by fresh medium and the cells were heat-shocked at 43° C. for 3 hours, then returned to 37° C. (22). After two days of incubation at 37° C., transfected cells were transferred onto monolayers of Vero, CEF or MRC5 cells and incubated at 37° C. in 10 cm dishes, except for CEF which were incubated at 32° C. Fluorescent cells appeared rapidly after 2-3 days of co culture on Vero, CEF or MRC5 cells. Infected cells expanded rapidly in focuses. The recombinant virus was highly syncytial in Vero cells and non-syncytial on CEF and MRC5 cells. Single syncytia or infectious focuses were transferred to 35 mm wells of Vero, CEF or MRC5 cells, then expanded to larger dishes by adding fresh cells. Virus was harvested from CEF or MRC5 cells after 5 days of infection, and from Vero cells when syncytia involved 80-90% of the culture (usually after 2 days) by scraping infected cells, freeze-thawing of cells and medium, and centrifugation to remove cellular debris. Such viral productions were titrated using the TCID₅₀ titration method. Briefly, Vero cells were seeded into 96-well plate (7500 cells/well) and infected by serial 1:10 dilutions of virus sample in DMEM/5% FCS. After incubation at 37° C. for 7 days, cells were stained with crystal violet and the virus dilution that resulted in infection in 50% of test unit was determined. The 50% end point described as tissue culture infectious dose (TCID₅₀) was calculated by the Kärber method (3). Recombinant virus rescued and grown on Vero cells had titers of 10⁷-10⁸ TCID₅₀/ml, and virus rescued and grown on CEF or MRC5 cells had lower titers of 10⁴-10⁶ TCID₅₀/ml.

The invention provides the technology for the construction and production of recombinant vectors, especially Human Immunodeficiency Virus (HIV)-TRIP lentiviral vectors, expressing the T7 RNA polymerase and measles Schwarz N and P proteins under the control of the cytomegalovirus (CMV) promoter. These vectors can be used to efficiently transduce in vitro at high level almost all cells, particularly human or mammalian cells. Such cells can be used as helper/transcomplementary cells able to generate de novo recombinant measles viruses after transfection by full-length infectious viral antigenomic cDNA or by modified cDNA clones as defined above.

The present invention allows rescuing any non-segmented negative-strand RNA viruses, such as measles viruses, from cDNA, optionally modified, without contamination by any other helper virus such as vaccinia virus, Due to the high transduction efficiency of lentiviral vectors, this method allows to generate cells expressing a very high level of helper proteins. Because retrovirus-based recombination, particularly lentiviral-based recombination, of foreign DNA into chromosomic DNA is genuine as compared to the illicit plasmid-based recombination, the helper cells generated by this method are very stable and their high efficiency to rescue non-segmented negative-strand RNA viruses from cDNA is maintained after multiple serial passages.

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1-68. (canceled)
 69. A method to produce an infectious non-segmented negative-strand RNA virus, comprising: a) providing a cell line comprising: i) a stably integrated retroviral vector comprising a coding sequence for an RNA polymerase; ii) a stably integrated retroviral vector comprising a coding sequence for a nucleoprotein (N) of a non-segmented negative-strand RNA virus; and iii) a stably integrated retroviral vector comprising a coding sequence for a polymerase cofactor phosphoprotein (P) of a non-segmented negative-strand RNA virus; b) transfecting the cell line of a) with (i) a vector comprising the coding sequence for a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus, and (ii) a vector comprising a cDNA clone of a non-segmented negative-strand RNA virus; c) combining transfected cells of b) with cells competent to sustain the replication and production of the non-segmented negative-strand RNA virus to form a co-culture; and d) recovering the infectious non-segmented negative-strand RNA virus from the co-culture.
 70. The method of claim 69, wherein the N, P, and L proteins are from the same type of non-segmented negative-strand RNA virus.
 71. The method of claim 69, wherein the N, P, and L proteins are from a measles virus.
 72. The method of claim 71, wherein the measles virus is a Schwarz strain measles virus.
 73. The method of claim 69, wherein the cDNA clone of a non-segmented negative-strand RNA virus is a cDNA clone of a measles virus.
 74. The method of claim 73, wherein the measles virus is a Schwarz strain measles virus.
 75. The method of claim 71, wherein the cDNA clone of a non-segmented negative-strand RNA virus is a cDNA clone of a measles virus.
 76. The method of claim 75, wherein the measles virus is a Schwarz strain measles virus
 77. The method of claim 69, wherein the competent cells in c) are Vero (African green monkey kidney) cells, CEF (chick embryo fibroblast) cells or MRC5 cells.
 78. The method according to claim 69, wherein the nucleotide sequence of the cDNA clone of the measles virus is modified by insertion, at a permissive site, of at least one heterologous nucleic acid.
 79. The method according to claim 78, wherein the at least one heterologous nucleic acid encodes at least one epitope.
 80. A method to produce an infectious non-segmented negative-strand RNA virus, comprising: a) providing a cell line comprising: i) a stably integrated retroviral vector comprising a coding sequence for an RNA polymerase; ii) a stably integrated retroviral vector comprising a coding sequence for a nucleoprotein (N) of a non-segmented negative-strand RNA virus; iii) a stably integrated retroviral vector comprising a coding sequence for a polymerase cofactor phosphoprotein (P) of a non-segmented negative-strand RNA virus; and iv) a stably integrated retroviral vector comprising a coding sequence for an RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus; b) transfecting the cell line of a) with a vector comprising a cDNA clone of a non-segmented negative-strand RNA virus; c) combining transfected cells of b) with cells competent to sustain the replication and production of the non-segmented negative-strand RNA virus to form a co-culture; and d) recovering the infectious non-segmented negative-strand RNA virus from the co-culture.
 81. The method of claim 80, wherein the N, P, and L proteins are from the same type of non-segmented negative-strand RNA virus.
 82. The method of claim 80, wherein the N, P, and L proteins are from a measles virus.
 83. The method of claim 82, wherein the measles virus is a Schwarz strain measles virus.
 84. The method of claim 80, wherein the cDNA clone of a non-segmented negative-strand RNA virus is a cDNA clone of a measles virus.
 85. The method of claim 84, wherein the measles virus is a Schwarz strain measles virus.
 86. The method of claim 82, wherein the cDNA clone of a non-segmented negative-strand RNA virus is a cDNA clone of a measles virus.
 87. The method of claim 86, wherein the measles virus is a Schwarz strain measles virus
 88. The method of claim 80, wherein the competent cells in c) are Vero (African green monkey kidney) cells, CEF (chick embryo fibroblast) cells or MRC5 cells.
 89. The method according to claim 80, wherein the nucleotide sequence of the cDNA clone of the measles virus is modified by insertion, at a permissive site, of at least one heterologous nucleic acid.
 90. The method according to claim 89, wherein the at least one heterologous nucleic acid encodes at least one epitope. 